Sustained signaling by Ras-family GTPases can lead to cancer as well as developmental disorders. Recent findings reveal that H- and K-Ras are monoubiquitinated and that these modified proteins accumulate in the GTP-bound (activated) state in cells. Our preliminary data reveal that monoubiquitinated Ras is refractory to GTPase activating proteins, but is otherwise fully functional. Based on these findings, we propose that monoubiquitination activates Ras directly, in the absence of a sustained hormone stimulus or oncogenic mutation. Here we propose a comprehensive (in vivo and in vitro) analysis of Ras monoubiquitination in yeast. Aim 1. Functional analysis of monoubiquitinated Ras1. We have shown recently that Ras1, but not Ras2, is monoubiquitinated in yeast. This provides a unique opportunity to compare two functionally similar proteins that are regulated differently. Our hypothesis is that monoubiquitination leads to sustained activation of Ras1. We will identify the structural determinants for selective ubiquitination of Ras1. Using ubiquitination-deficient mutants we will determine how this modification affects Ras signaling in vivo. To establish mechanism, we will determine how monoubiquitination affects the biochemical properties of the protein and its binding partners in vitro. Aim 2. Dynamic regulation of Ras1 monoubiquitination. Our hypothesis is that Ras1 monoubiquitination is a dynamically regulated event. Our preliminary evidence indicates that Ras1 is monoubiquitinated as part of a stimulus-dependent, phosphorylation-dependent feedback mechanism. Using available gene deletions we will determine which enzymes are necessary for the phosphorylation, monoubiquitination and deubiquitination of Ras1. Using purified proteins we will establish which enzymes are sufficient for each modification in vitro. Aim 3 Downstream targets of monoubiquitinated-Ras1. The yeast genome encodes 43 ubiquitin-binding domain (UBD) proteins, which in many cases serve as intracellular ubiquitin receptors. Our hypothesis is that select UBD proteins interact directly and specifically with monoubiquitinated Ras1, and thereby alter Ras1 trafficking and signaling functions. Using purified proteins and available gene deletion mutants, we will identify the UBDs that target monoubiquitinated Ras1, but not unmodified Ras1 or Ras2. Our approach integrates powerful genomics and proteomics tools, many of which are available only in yeast, to study a process that has clear relevance to human health and disease. If successful, our experiments will reveal (for the first time) proteins responsible for the initiation, addition, removal, and recognition of ubiquitin bound to Ras. Yeast employ a Ras signaling apparatus analogous to that found in humans. Thus a fuller understanding of how Ras signaling is modulated in yeast could eventually lead to fundamentally new approaches to treat Ras-related disease in humans.